A tandem repeat in DNA represents two or more contiguous approximate copies of a pattern of nucleotides. Tandem repeats have been shown to be associated cause a variety of human diseases. Dramatic expansion of trinucleotide repeats has been associated with such diseases as fragile-X mental retardation (see Verkerk, et al., (1991) Cell, 65, 905-914), Huntington's disease (see Huntington's Disease Collaborative Research Group. (1993) Cell, 72, 971-983), myotonic dystrophy (see Fu, et al., (1992) Science, 255, 1256-1258), spinal and bulbar muscular atrophy (see La Spada, et al., (1991) Nature, 352, 77-79) and Friedreich's ataxia (see Campuzano, et al., (1996) Science, 271, 1423-1427).
Fragile X syndrome is one of the most common causes of inherited mental retardation, occurring in approximately one in 1,250 males and approximately one in 2,500 females. Males with fragile X syndrome typically exhibit some degree of mental impairment, ranging from learning disabilities to mental retardation to autism. Characteristic physical features (e.g., enlarged ears, elongated face with prominent chin), connective tissue problems (e.g., mitral valve prolapse, and double-jointed fingers), and characteristic behaviors (e.g., attention deficit disorders, speech disturbances, and unusual responses to various touch, auditory, or visual stimuli) may also be exhibited. Affected females present with similar but milder mental impairment, physical characteristics, and behavioral characteristics as those of affected males.
The mutation responsible for fragile X syndrome involves expansion of a trinucleotide (CGG) tandem repeat sequence located in the 5′ untranslated region of the FMR1 gene on the X chromosome. The number of CGG repeats in the FMR1 gene determines whether an individual is normal or has one of the two categories of mutation: premutation and full mutation. The number of repeats ranges from less than 55 repeats in normal, non-carrier individuals, whereas a premutation consists of 55 to 200 repeats and full mutation consists of more than 200 repeats (Chen et al. Hum. Mol. Genetics 12(23):3067-74, 2003).
Both males having a premutation and females having a premutation in one FMR1 gene are carriers but are unaffected. Male carriers are referred to as “normal transmitting” males, and pass on the mutation, relatively unchanged in size to each daughter. Although such daughters are unaffected, they are at risk of having affected offspring because a premutation is susceptible to expansion after passage through a female meiosis. Furthermore, the larger the premutation, the higher the risk of expansion to a full mutation in any offspring.
Most males with a full mutation exhibit mental retardation and stereotypical physical and behavioral characteristics. For females with a full mutation in one FMR1 gene, about one-third exhibit normal intelligence, about one-third exhibit borderline intelligence, and about one-third exhibit mental retardation.
Currently, the industry standard for screening for carriers or affected individuals with expansion of tandem repeat regions such as Fragile X is a combination of PCR amplification of the tandem repeat region and analysis by Southern blotting.